Cloning sticky PCR products
svetlov at oncology.wisc.edu
Thu Apr 16 12:32:24 EST 1998
In article <35353127.D096AE5 at wadsworth.org>, Brian Cohen
<cohen at wadsworth.org> wrote:
> I haven't had a chance to check out the Biotechniques article yet, but
> I've had rather good luck cloning PCR products, as small as 125 bp and up to
> 3kb. I've found that PCIA extraction followed by restriction digest and
> agarose gel purification works pretty well. We've been using the Geneclean
> kit (BIO101, no relation to the author) even for the very small PCR products
> with good success. I've never tried to quantitate the efficiency of this
> process, although I know that skipping the PCIA extraction usually results
> in no digestion.
> Hope this helps, I look forward to seeing other protocols as well.
As long as the PCR product contains enough nt's on the end for enzymes to
cut I never had a problem cloning PCR products into vectors with cohesive
ends - same sites or compatible ends (e.g. SalI into XhoI). Efficiency is
routinely >95% and I'm getting yeilds good enough not just for cloning but
for randomly mutagenized library construction. After amplification I purify
PCR products in agarose gel, transfer onto DE81 paper elute and either
PCI-precipitate or clean-up with Promega kit (recently). Digestion done
overnight, vector dephosphorylated and used in 10-20 fold molar excess to
the insert. No concatemirization detected yet by sequencing. I understand
that for some people doing a gel purification is a hassle but it does save
time overall. Couple of times I did simulpurification/cloning of 18
different PCR products and did not even crosscontaminate any. Call me
old-fashioned but this shit works like a swiss clock.
Vladimir Svetlov, Ph. D.
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
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